Adenosine is considered to be a hormone with localized effects on most cells of higher organisms. Despite the widespread and important physiological and pathological effects of adenosine, agonists and antagonists of the adenosine receptors have not been widely exploited for pharmaceutical purposes. The long-range objective of this program is the rational design of adenosine receptor-specific drugs. Toward this end the current research is designed to determine the adenosine and xanthine binding-pockets of the A1- and A2-adenosine receptors and to understand the spatial arrangement of these receptors. These receptors have been cloned and will be over- expressed to obtain sufficient material to study the receptors at the molecular level. Specifically, this application proposes to map the adenosine receptors by affinity-labeling several locations within the ligand binding sites and identifying subsequently generated fragments of the receptors. Photoaffinity and affinity probes based on the structures of both agonists and antagonists will be designed to label the ribose- binding domain, the N6-aralkyl-binding domain and the 2-aralkoxy-binding domain of agonists and the domain for the 8-cycloalkyl moiety of potent xanthine antagonists. In addition, agents will be used that should help identify the purine-binding pocket.